Dysfunction of vitamin B12 metabolism can produce a form of cobalamin deficiency which has as its clinical consequences methylmalonicacidemia, homocystinuria, mental retardation and megaloblastic anemia. The purpose of this program is to identify and characterize the enzymes that catalyze the conversion of aquocobalamin and methylcobalamin to the principal coenzyme form of the vitamin, adenosylcobalamin. These studies will employ a rat liver model which is available in both normal and B12-deficient forms. Subcellular fractionation of rat liver will be used to localize enzyme activities and facilitate their purification by affinity chromatographic techniques. Parallel studies will be conducted on the cobalamin auxotroph Lactobacillus leichmannii, which mimics mammalian cell metabolism of the vitamin. The microbial model is expected to be a convenient source of: 1. cobalamin reductase(s) and adenosylating enzyme, enzymes that are involved in adenosylcobalamin biosynthesis; and, 2. a demethylase which is believed to be responsible for the first step in the conversion of methylcobalamin to adenosylcobalamin. Regenerating rat liver and rat hepatomas will be used for studying cobalamin coenzyme metabolism during the proliferative state in mammalian cells. Cobalamin analogs, designed to block adenosylcobalamin formation, will be synthesized and tested against purified enzyme components of the metabolic pathway and against proliferating cells.